Efficient Stable Expression of Nuclear H5N1 Avian Influenza Virus HA2 Transgene in Chlamydomonas reinhardtii

Green biotechnology is the future of biopharmaceuticals production. The use of algae to produce biopharmaceuticals is of interest in vaccine-based control programs because of cost and environmental safety considerations. An attempt was made to express avian influenza virus (AIV) immunogens in algae because the virus is a serious economic, veterinary and public health threat. A commercial system was modified to allow expression of H5N1 AIV hemagglutinin subunit 2 (HA2) in the microalga Chlamydomonas reinhardtii. Codon-optimized AIV H5N1 HA2 (coHA2) sequences were synthesized and cloned into the transfer vector pChlamy_3/D-TOPO® (3DcoHA2). 3DcoHA2 plasmids were used to transform C. reinhardtii strain cc-125 by electroporation. Proper nuclear integration was confirmed in 16% of screened transformants selectively amplified in Hygromycincontaining TAP media. coHA2 mRNA transcription was confirmed using RT-PCR. AIV HA2 expression was confirmed using Western Blot analysis utilizing mono-specific AIV H5N2 polyclonal chicken antisera. Expressing transformants were maintained on Hygromycin-containing TAP agar for 26 weeks (15 subcultures). Expressing transformants maintained cell shape, motility and, growth characteristics similar to non-transformed C. reinhardtii cc-125. A coHA2-C terminus GFP was used to visualize HA2 expression in vivo using confocal microscopy. Background-normalized GFP-specific fluorescence of transformants was 15 % of the total cellular fluorescence. Fluorescence in GFP channels 508, 518, 528 and 538 nm was 3.9% of the total cellular fluorescence of non-transformed algae. Taken together, results indicate efficient stable expression of the AIV HA2 transgene and, warrant further investigation into immunogenic potential of the algae-expressed HA2.

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